Proteoglycans are glycoproteins formed of saccharide chains (glycosaminoglycans) covalently attached to proteins (non-patent document 1). Glycosaminoglycans are polysaccharides typically composed of 40-100 repeating disaccharide units characterized in that they are sulfated to various degrees. Glycosaminoglycans include chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, etc. Among others, heparan sulfate-containing proteoglycans known as heparan sulfate proteoglycans (HSPGs) include syndecans and glypicans expressed on the plasma membrane, and perlecan and agrin secreted in the basement membrane. In mice, heparan sulfate is expressed highly in lung and kidney, and weakly in skeletal muscle, liver, skin and brain (non-patent document 2). HSPGs are known to influence the activity of growth factors and to participate in the growth and differentiation of cells. For example, fibroblast growth factors, heparin-binding epidermal growth factor-like growth factor and amphiregulin transduce signals into cells via receptors of these growth factors as they bind to heparan sulfate (non-patent documents 3, 4, 5, 6). In cancer, HSPGs were also reported to participate in the growth and metastasis of cancer cells (non-patent documents 7, 8).
Heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) is an enzyme that adds a sulfate group to the 6-O position of glucosamine that constitutes heparan sulfate. Known similar enzymes (sulfotransferases) include HS6ST1 and HS6ST3 (non-patent document 9). These members of the HS6ST family are type II membrane proteins localized in the Golgi in cells and act as enzymes (non-patent document 10). HS6ST1 is secreted outside cells as well upon cleavage near the transmembrane domain (non-patent documents 11, 12, 13). HS6ST2 was also suggested to be secreted extracellularly (non-patent document 14). In fact, mouse HS6ST2 (mHS6ST2) is secreted extracellularly as well when it is forcibly expressed in CHO cells (non-patent document 15). Although the secretory mechanism is unknown, the following evidence exists: mHS6ST2 remaining in the Golgi and secreted mHS6ST2 have the same molecular weight; the N-terminal region of mouse HS6ST3 including the transmembrane domain may be cleaved as a signal peptide; and a variant of mHS6ST2 containing an N-terminal extension of 146 amino acids is not secreted extracellularly (non-patent document 15). Generally, it is thought that extracellularly secreted HS6ST2 does not act as an enzyme because the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate is rapidly degraded in blood.
The steric structure of HS6ST2 has not been elucidated, but considered to recognize and bind to partial sequences of heparan sulfate of up to six saccharide chains (non-patent document 16). On the other hand, the crystal structure of mouse HS3ST1, i.e., an enzyme that adds a sulfate group to the 3-O position of glucosamine of heparan sulfate has been analyzed, and it has been shown to bind to heparan sulfate with micromolar affinity (Kd=2.79 μM) (non-patent document 17). Extracellularly secreted HS6ST2 also seems to be able to bind to heparan sulfate on the plasma membrane. Membrane-associated HSPGs are expressed in almost all cells. The expression level is about 105-106 molecules per cell, and they are mostly taken up by cells with a half-life of 3-8 hours and degraded in lysosomes (non-patent document 18). Indeed, it is known that peptides such as HIV-Tat and bFGF; nucleic acids such as polylysine-DNA complexes; polyamines or anti-HSPG antibodies are taken up by cells via HSPGs (non-patent documents 19, 20, 21).
Thus, it is presumed but has not been verified that secreted HS6ST2 bound to HSPGs is also taken up by cells. Physiological effects of anti-HS6ST2 antibodies and their applications for pharmaceutical uses have not been verified, either.